An important question in muscle structure, debated for the past two decades and still unresolved, is whether a third kind of filament - a continuous one (distinct from the discontinuous actin and myosin filaments) - exists within myofibrils of striated muscle. During the first two years of our initial grant, we discovered a group of three extremely large proteins (Mr greater than 500,000) in a wide range of vertebrate and invertebrate striated muscles. These proteins, which we have named "titins" because of their titanic size, differ in solubility properties and locations within myofibrils from thin and thick filament associated proteins. Interestingly, titins can, under a variety of conditions, form birefringent filamentous aggregates which appear to contain ultrathin filaments (20A diameter). Furthermore, filaments of similar size have been detected in myofibril residues after most thin and thick filaments have been extracted away. Based on these results, we suggested that titins may be components of the putative longitudinal filaments in striated myofibrils. We propose to continue this investigation. Specifically we propose (1) to purify and characterize each of the three titins from rabbit skeletal muscle; (2) to localize titins in myofibrils and striated muscle fibers with immunocytological methods; (3) to search for ultrathin filaments by resolving myofibrils into component structures using selective extraction and fractionation procedures; (4) to search for manifestations of possible control mechanisms, in particular, phosphorylation and calcium ion binding; and (5) to perform a survey of the distribution and organization of titins in nature. We hope that the biochemical and structural studies proposed here will lead to better understanding of the structure and function of contractile apparatus, the mechanical properties of striated muscle and the mechanism of muscular contraction.